Phenacetin (PEAA) is converted by two different mechanisms to a reactive metabolite that is similar, if not identical to the reactive metabolite of acetaminophen (PHAA). 1) Incubation of hamster liver microsomes with PEAA, NADPH, and glutathione (GSH) in the presence of a 1802 atmosphere led to a 50% incorporation of 18O into the para position of the PHAA-GSH conjugate, but no 18O was incorporated into the conjugate when p-18O-PHAA was incubated with the enzyme system. 2) When p-18O-PEAA was incubated with the system in air, there was a 50% loss of 18O in the PHAA-GSH conjugate, but no 18O was lost when acetaminophen was incubated in the system. 3) When the N-O-glucuronide of PEAA was incubated in pH 7.4 buffer in H218O, the 18O was incorporated into the para position of the GSH conjugate. Thus there are three pathways for the formation of the reactive metabolite from PEAA. However, the mercapturates excreted in the urine after administration of 18O-PHAA-PEAA showed no loss of 18O in the metabolite derived from PHAA and only a 15% loss of 18O in the metabolite derived from PEAA. Thus at least 70% of the mercapturate formed after the administration of PEAA was formed by way of PHAA.